Regulation of translation rate is of crucial importance for such downstream co-translational pro- cesses as protein folding and modification, ligand binding, oligomerization, and interactions with chaperones and membranes. Although specific elements that modulate translation rate are well known, at present there is virtually a complete lack of results describing how such elements quantitatively affect translation rate. Our overall goal is to address this lack by quantifying the modulation of the rate of translation by E. coli ribosomes of specific mRNA and nascent peptide sequences, and to elucidate the mechanisms of such modulation. We use an approach coupling Total Internal Reflection Fluorescence Microscopy (TIRFM) with single molecule Fluorescence Resonance Energy Transfer (smFRET). Fluorescent reporter and smFRET pairs are introduced specifically into both aminoacyl tRNAs and ribosomal proteins L1 and L11, which are proximal to the tRNA E-site and A-site, respectively. The availability of these fluorescent-labeled compo- nents of the protein synthesis machinery permits observation on single ribosomes and in real time of FRET interactions monitoring aminoacyl-tRNA binding to the ribosomal A-site, interme- diate motions of tRNAs in adjacent sites, translocation of the tRNAs to the P- and E-sites, and release of discharged tRNA from the E-site. Single molecule assays have been developed both for short model mRNAs that emphasize pairing with fluorescent tRNAs and for mRNAs coding full-length proteins with the natural complement of all amino acids. Full expression of a rapidly maturing variant of green fluorescent protein, Emerald GFP (EmGFP), is signaled by fluores- cence of single EmGFP molecules in the TIRF microscope. Together, these approaches allow both discrimination of individual steps within the elongation cycle and continuous kinetic profiles of mRNA translation, providing unique insights into the regulation of translation rates in prokary- otic protein synthesis not accessible from other techniques. Our results will allow us to quantify the effects of modulatory elements and to determine if new elements and synergistic or antago- nistic effects are operative. Crucial for the correct interpretation of our results is the performance of control experiments demonstrating that the rate effects we measure result from introduction of the intended modulating element, rather than from involvement of one or more other ele- ments. We have 3 Specific Aims that focus on determining how the rhythm of protein synthesis is modulated by: 1. Codon usage and codon pair usage; 2. Pause-inducing nascent peptides bound within the peptidyl transferase center of the ribosomes and the peptide exit tunnel; and 3. Pausing elements present in combination. Overall, our program of studying several pause ele- ments at the full protein expression level and within individual elongation cycles will define the magnitudes and detailed mechanisms of translation regulation.